Newborn Blood Cells Research Articles

Delayed Platelet Recovery Following Cord Blood Transplantation: Insights from a Mouse Model.

Umbilical cord blood (CB) is an important source of hematopoietic stem cells for stem cell transplantation and is being used with increasing frequency. A major concern related to clinical CB transplantation is the long delay in platelet recovery. Megakaryopoiesis is characterized by the acquisition of lineage-specific markers (e.g. CD41) during the early stages that is followed by polyploidization (DNA content > 4N) during the later stages of megakaryopoiesis. Using a newborn blood (NB) model of CB transplantation that was previously developed in our laboratory, we asked whether the delay in platelet recovery is a result of a decrease in the rate of megakaryocyte production or a delay in their maturation. C57BL/6 mice were transplanted with either murine adult bone marrow (BM) cells or murine new-born blood (NB) cells following lethal irradiation. We had previously shown that the concentration of Lin-Sca-1+c-Kit+ stem cells in murine adult BM was approximately 3 times higher than that in NB. To correct for this difference in stem cell concentration, irradiated mice were transplanted with either 0.5 ×106 BM cells or 2×106 NB cells. Platelet counts at 2 and 4 weeks were lower in mice transplanted with NB cells than in mice transplanted with BM cells (Table 1). Interestingly, the platelet counts became comparable in NB and BM recipients at 8 weeks post-transplantation. We compared the ability of BM cells from both NB and BM recipients to form CFU-Meg colonies in methylcellulose. At 2 and 4 weeks post-transplantation, BM cultures derived from NB recipients generated fewer CFU-Meg colonies than cultures from BM recipients (Table 1). Interestingly, after 8 weeks, the numbers of CFU-Meg from both stem cell sources were similar. We also compared the ability of BM cells from NB and BM recipients to differentiate into megakaryocytes in liquid culture, using CD41 expression and polyploidy as markers of megakaryocytic maturation. At 2 weeks post-transplantation, cultures from NB recipients generated 13% CD41+ cells whereas cultures from BM recipients generated 22% CD41+ cells. However, by 4 weeks post-transplantation, the numbers of CD41+ cells were similar in cultures derived from NB recipients and BM recipients (Table 1). Moreover, at 2 and 4 weeks post-transplantation, there were fewer polyploid cells in liquid cultures from NB recipients compared to BM recipients (Table 1). The lower number of polyploid cells was commensurate with the lower number of CD41+ cells. This suggests that the rate of maturation of megakaryocyte is similar in NB and BM. In conclusion, our studies show that CB stem cells generate megakaryocytic progenitors at a slower rate than BM stem cells and that the delay in platelet recovery is not a result of a delay in the maturation of megakaryocytic progenitors. Thus, in order to increase the rate of platelet recovery following CB transplantation, the focus should be on enhancing the rate of production of megakaryocytic progenitors from hematopoietic stem cells.Table 1Platelet(×103/μl)CFU-Meg(Colonies/1 × 105 Cells)CD41(%)Ploidy(% >4N DNAContent)normalBM(n=5)1200±12035.5±555±527±32 weeksNBT-2M(n=5)190± 3810±213±27.5±1BMT-0.5M(n=5)400±5017±422±111±24 weeksNBT-2M(n=5)550±5918±226±214.5±2BMT-0.5M(n=5)730±11023±327±118±38 weeksNBT-2M(n=5)930±11539±7N/AN/ABMT-0.5M(n=5)970±13040±4N/AN/A

Graft-Versus-Host Disease and Graft-Versus-Leukemia Effect in Mice Grafted With Peripheral Newborn Blood

We previously used peripheral newborn blood (NBB) as a possible in vivo experimental model for cord blood (CB) transplantation and showed that B10.D2 NBB cells successfully reconstituted adult (DBA/2 × B10.D2)F1 mice without causing graft-versus-host disease (GVHD), probably because of their phenotypic and functional immaturity. Here we investigated the influence of T-cell maturation occurring in NBB cells during the early postbirth period on the degree of engraftment, the incidence of GVHD, and the graft-versus-leukemia (GVL) potential. These parameters were compared in recipients grafted with bone marrow (BM) cells. We observed an increased percentage of CD4+ mature T cells accompanied by the acquisition of proliferative responses to phytohemagglutinin (PHA) and to allogeneic cells of day-5 NBB cells. The capacity of day-2 NBB to engraft was moderately reduced and recipients developing GVHD were occasionally observed after the graft of day-5 NBB cells. No GVL effect was evidenced regardless of the time of postbirth blood collection. However, the GVL effect can be obtained by the delayed infusion of donor mature T cells to recipients grafted with day-0 NBB, without causing GVHD. In contrast, the same protocol applied to mice grafted with BM cells induced GVHD mortality of all recipients. Interleukin (IL)-10 but not IL-2 messenger RNA was expressed in NBB cells as opposed to BM cells. These findings suggest that, in terms of GVHD incidence, delayed infusion of mature T cells as post-transplant tumor immunotherapy would be more effective when applied after CB than after BM transplantation.

Graft-Versus-Host Disease and Graft-Versus-Leukemia Effect in Mice Grafted With Peripheral Newborn Blood

We previously used peripheral newborn blood (NBB) as a possible in vivo experimental model for cord blood (CB) transplantation and showed that B10.D2 NBB cells successfully reconstituted adult (DBA/2 x B10.D2)F1 mice without causing graft-versus-host disease (GVHD), probably because of their phenotypic and functional immaturity. Here we investigated the influence of T-cell maturation occurring in NBB cells during the early postbirth period on the degree of engraftment, the incidence of GVHD, and the graft-versus-leukemia (GVL) potential. These parameters were compared in recipients grafted with bone marrow (BM) cells. We observed an increased percentage of CD4(+) mature T cells accompanied by the acquisition of proliferative responses to phytohemagglutinin (PHA) and to allogeneic cells of day-5 NBB cells. The capacity of day-2 NBB to engraft was moderately reduced and recipients developing GVHD were occasionally observed after the graft of day-5 NBB cells. No GVL effect was evidenced regardless of the time of postbirth blood collection. However, the GVL effect can be obtained by the delayed infusion of donor mature T cells to recipients grafted with day-0 NBB, without causing GVHD. In contrast, the same protocol applied to mice grafted with BM cells induced GVHD mortality of all recipients. Interleukin (IL)-10 but not IL-2 messenger RNA was expressed in NBB cells as opposed to BM cells. These findings suggest that, in terms of GVHD incidence, delayed infusion of mature T cells as post-transplant tumor immunotherapy would be more effective when applied after CB than after BM transplantation.

Newborn Blood Can Engraft Adult Mice Without Inducing Graft-Versus-Host Disease Across Non H-2 Antigens

Cells derived from human cord blood were recently used instead of bone marrow (BM) for transplantation. However, several questions concerning the potential of these cells to reconstitute the hematopoietic and immunologic system of the recipient and to induce a graft-versus-host disease (GVHD) remain unanswered. Here we used newborn blood (NBB) cells from B10.D2 mice to engraft lethally irradiated (DBA/2 x B10.D2)F1 recipients incompatible for multiple non H-2 antigens. Few mature T cells were found in NBB as in adult BM and both contain around 10% to 20% SCA-1+ pluripotent progenitor cells. Yet numerous immature CD4+CD8+ TCR alpha/beta low Thy-1high T cells were present in NBB. In contrast, adult peripheral blood (PB) exhibits a mature T-cell phenotype and contains few progenitor cells. The injection of blood pooled from one to three newborn mice resulted in the engraftment of 71% to 86% of F1 recipients, which survived more than 100 days without clinical signs of GVHD. The injection of BM leads to 100% survival whereas the injection of adult PB resulted in rapid mortality, both without signs of GVHD. In NBB-engrafted F1 surviving mice, T-cell reconstitution preceded B-cell reconstitution, and the degree of donor cell chimerism increased progressively with time. Additionally, 2 to 4 months after transplantation, T cells from these mice were tolerant to host non H-2 antigens but kept reactivity against third-party Ags. Host specific tolerance in NBB-engrafted F1 mice was confirmed by in vivo transfer experiments. In conclusion, NBB cells were able to reconstitute the lymphohematopoietic system of lethally irradiated adult mice without inducing GVHD against incompatible non H-2 antigens. Thus, this experimental model in vivo may be relevant to the human cord blood transplantation.

Newborn blood used as a source of donor cells in a murine model of transplantation across non-MHC antigens.

Cells derived from human cord blood instead of bone marrow were recently used for transplantation. However, several questions concerning the potential of this source of cells to reconstitute the hematopoietic and immunologic system of the recipient and to induce graft-versus-host disease (GVHD) remain unanswered. We used newborn blood (NBB) cells from B10.D2 mice to engraft lethally irradiated (DBA/2 x B10.D2)F1 recipients incompatible for multiple non-H-2 antigens. The median volume of NBB collected from one mouse ranged between 40 and 50 microliters and the number of nucleated cells was approximately 4-5 x 10(5) per sample. We first established that NBB contains around 10-20% of stem cells (SCA-1+) and 30% of CD4+CD8+ Thy-1+ immature T cells. The injection of blood pooled from one to three newborn mice resulted in engraftment of 71%-86% of F1 recipients that survived more than 100 days. Long-term surviving mice exhibited mixed chimerism (approximately 69% of cells of donor origin) 2-4 months after transplantation, and clinical signs of GVHD across minor histocompatibility Ags (mHAgs) were never observed. Additionally, mixed lymphocyte reaction and cytotoxic assay responses of those mice against host antigens were undetectable, while reactivity against unrelated H-2 Ag was normal. The establishment of host-specific tolerance in NBB engrafted mice was confirmed by in vivo transfer experiments. In conclusion, NBB cells reconstituted the lymphohematopoietic system of lethally irradiated mice without inducing GVHD. However, the question of the presence of an active antileukemic effect remains to be answered.